ABSTRACT: Limited research has suggested iron oxide nanoparticles (FeNP) have an inhibitory effect against several different genera of bacteria: Staphylococcus, Bacillus and Pseudomonas spp. In this study we looked at the effect of three different sets of Fe3O4 nanoparticles (FeNPs) on the development of Pseudomonas aeruginosa PAO1 biofilms. Two of the tested NPs were SPIONs (Superparamagnetic Iron Oxide Nanoparticles). Exposure of cells to the SPIONs at concentrations up to 200 μg/ml resulted in an increase in biofilm biomass by 16 h under static conditions and a corresponding increase in cell density in the bulk liquid. In contrast, these biofilms had decreased levels of extracellular DNA (eDNA). Fe(II) levels in the supernatants of biofilms formed in the presence of FeNPs exceeded 100 μM compared with 20 μM in control media without cells. Spent cell supernatants had little effect on Fe(II) levels. Cells also had an effect on the aggregation behavior of these nanoparticles. SPIONs incubated with cells exhibited a decrease in the number and size of FeNP aggregates visible using light microscopy. SPIONs resuspended in fresh media or spent culture supernatants formed large aggregates visible in the light microscope upon exposure to a supermagnet; and could be pelleted magnetically in microtitre plate wells. In contrast, SPION FeNPs incubated with cells were unaffected by exposure to the supermagnet and could not be pelleted. The results of this study indicate a need to reconsider the effects of FeNPs on bacterial growth and biofilm formation and the effect the bacterial cells may have on the use and recovery of SPIONs.